Composition comprising HMSs/HMOs and use thereof

ABSTRACT

The application relates to a method for the prophylaxis or treatment of non-infectious diarrhea in a human, the method comprising administering to the human an effective amount of one or more human milk oligosaccharides.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a continuation-in-part application of and claims priority to,U.S. application Ser. No. 15/147,112 entitled “COMPOSITION COMPRISINGHMSs/HMOs AND USE THEREOF” and filed on May 5, 2016 for Thierry Hennetet al. and to U.S. patent application Ser. No. 15/034,593 entitledSYNTHETIC COMPOSITION AND METHOD FOR TREATING IRRITABLE BOWEL SYNDROMEand filed on May 5, 2016 for Thierry Hennet et al., and claims thepriority to PCT/DK2015/050332 entitled SYNTHETIC COMPOSITION AND METHODFOR TREATING IRRITABLE BOWEL SYNDROME and filed on Oct. 29, 2015 forThierry Hennet and claims priority to Denmark Application No. PC 20147063 which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions and methods employing human milkcomponents for the treatment of non-infectious diarrhea.

BACKGROUND Description of the Related Art

Diarrhea affects most individuals at some time during their lives. Itresults when the efficiency of the intestine for absorbing water,electrolytes, and nutrients is impaired. Approximately 8-9 litres offluid enters the intestines daily of which 1-2 litres represents foodand liquid intake, and the rest is from endogenous sources such assalivary, gastric, pancreatic, biliary, and intestinal secretions. Mostof the fluid, about 6-7 litres, is absorbed in the small intestine, andonly about 1-2 litres is presented to the colon. Most of this isabsorbed as it passes through the colon, leaving a stool output of about100-200 g/day. Therefore, small changes in this absorption efficiencycan radically change the wetness of the stool. A great variety of drugs,toxins, pathogens, and foodstuffs can impair the efficiency ofelectrolyte and water absorption, leading to diarrhea.

Normal bowel frequency ranges from three times a day to three times aweek in the healthy population. Diarrhea is the increased frequency ofstooling, with stool consistency less solid than normal. Acute \diarrheais defined as three or more stools per day of decreased form from thenormal, lasting for less than 14 days. If the duration of symptoms islonger than 1 month, it is considered chronic diarrhea. Most cases ofacute diarrhea are self-limited, caused by infectious agents (e.g.viruses, bacteria, parasites), and do not require medication unless thepatient is immunocompromised.

Mechanistically, absorption may be impaired by poorly absorbed,osmotically active solutes in the intestinal lumen, by alteration inabsorptive cell function, by increases in crypt cell secretion, and bytoo rapid transit of intestinal contents. Most often, absorption isimpaired by mechanisms acting in concert. For example, excessive volumeof intestinal contents can speed intestinal transit; cytokines fromepithelial inflammatory cells can enhance cryptal secretion, and caninfluence the enteric nervous system to speed transit; bile salts, andlong-chain fatty acids, malabsorbed in the small intestine, can blockwater and electrolyte absorption in the colon. The colon employs severalmechanisms to ensure it delivers to the recto sigmoid colon a formedstool, probably the most important factor in faecal continence. Thecolon has reserve capacity by which it can absorb 2-3 extra litres ofwater and electrolytes delivered from the small intestine in a day.Colonic bacteria ferment soluble carbohydrate and protein, which escapedsmall intestinal absorption, into absorbable gases and short-chain fattyacids. Otherwise, these unfermented, unabsorbable solutes would beosmotically active in colonic contents, and would cause diarrhea.

Most cases of diarrhea, especially acute diarrhea, are infectionrelated; especially due to Campylobacter, Salmonella, Shigella, E. coli,Vibrio and Aeromonas organisms. In most cases, the diarrhea resolvesitself within 48 hours and no treatment other than rehydration isneeded. Causes of non-infectious diarrhea include inflammatory boweldisease, irritable bowel syndrome, ischemic bowel disease, partial smallbowel obstruction, pelvic abscess in the recto sigmoid area, faecalimpaction, carcinoid syndrome, food allergies, lactose intolerance, theingestion of poorly absorbable sugars such as lactulose, acute alcoholingestion, and adverse effects of prescription medication; oftenantibiotics. Non-infectious diarrhea is often persistent, chronic anddifficult to treat. Common treatments include anti-motility agents likeLoperamide to reduce the number of stools, bile acid sequestrants,digestive enzymes containing lactase, probiotics, etc.

Non-infectious diarrhea is particularly common in irritable bowelsyndrome (IBS) patients. Irritable bowel syndrome is a clinicallyheterogeneous disorder of human patients, particularly adult, withchronic symptoms such as abdominal pain, abdominal discomfort, abdominalbloating, fatigue, and changes in bowel movement patterns, such aspatterns of loose or more frequent bowel movements, diarrhea andconstipation. Routine clinical tests on patients typically show noabnormalities, although their bowels may be more sensitive to certainstimuli, such as balloon insufflation testing. The worldwide prevalenceof IBS is about 10-20% (Longstreth et al. Gastroenterology 130, 1480(2006)) but may be higher in certain countries. The causes of IBS areunknown but disruptions of the brain-gut axis, acute gastrointestinalinfections, small intestinal bacterial overgrowths, antibiotic usagesand dysbiosis are thought to be important risk factors (Kim et al.Digest. Dis. Sci. 57, 3213 (2012)). Other risk factors are young age,prolonged fever, anxiety, and depression. Chronic low-grade inflammationcommonly occurs in IBS patients, but there are otherwise little or noobservable clinical manifestations.

Diagnosis of IBS is difficult. No biomarker-based tests can be performedto diagnose IBS. Diagnosis generally involves excluding conditions thatproduce IBS-like symptoms and then following a procedure to categorise apatient's symptoms. Ruling out parasitic infections, lactoseintolerance, and celiac disease is recommended for all patients before adiagnosis of IBS is made. Once diagnosed, patients are usuallyclassified in accordance with the Rome III criteria into four symptomsubtypes based on stool consistency: diarrhea predominant (IBS-D),constipation predominant (IBS-C), mixed subtype (IBS-M) with alternatingepisodes of both diarrhea and constipation, and unsubtyped IBS (IBS-U).

There is no cure for IBS and current treatments focus on attempting torelieve symptoms. Treatments take various forms such as dietaryadjustments, medication, and psychological interventions. Patienteducation and good doctor-patient relationships are also important.However, most treatment is unsatisfactory and most patients continue toexperience chronic pain, fatigue, and other symptoms. While IBS has nodirect effect on life expectancy, its high prevalence and significanteffects on quality of life make it a condition with a high social cost.The general hopelessness associated with IBS is a source of frustrationfor both patients and health care practitioners treating them.

Current research has implicated the gastrointestinal microbiota, thebrain-gut axis and the mast cells in the pathophysiology of IBS. Thehuman gastrointestinal microbiota includes at least 1,000 species ofbacteria, and about 10¹⁴ individual bacterial cells from about 160different species inhabit each individual's intestine (Qin et al. Nature464, 59 (2010)). It is believed that an individual's genetic make-up andacquired immunity, as well as environmental factors, influence theirgastrointestinal microbiota. The microbiota in turn shape theindividual's immunity and physiology within the gastrointestinal system.It is also believed that a healthy individual maintains a symbioticrelationship with the microbiota colonizing his/her intestines, while anindividual with IBS has an imbalance in this microbiota-hostinteraction.

Treatments that target gastrointestinal microbiota such as antibiotics,probiotics and prebiotics appear to alleviate the symptoms of IBS; atleast temporarily. For instance, the antibiotic rifaximin appears toreduce bowel movement in IBS-D patients.

Abdominal pain and discomfort associated with IBS is connected to thebrain-gut axis and the response to stress hormones. IBS patientstypically experience abnormal gut motility and visceral hypersensitivitymediated by the brain-gut axis or central stress response system. Onearm of the brain-gut axis is the central efferent pathway, which isformed by the sympathetic nervous system and thehypothalamic-pituitary-adrenal axis (HPA). In stress-sensitive disordersincluding IBS, stress hormones of the HPA axis, such asadrenocorticotropic hormone (ACTH), cortisol, and catecholamine arereleased. Some studies have shown that the HPA axis response in IBSpatients is caused by increased mucosal immune activation, which in turnincreases plasma cytokine levels to stimulate the HPA axis.

In addition to the gut microbiome and the gut-brain axis, the mast cellsmay also play an important role in the pathogenesis of IBS. Increasedmast cell infiltration and activation in distal gut segments areassociated with symptom onset and severity of IBS. These cells are alsoimplicated in the elevated response of visceral afferent nerves tomucosal stimulus in IBS patients. Mast cell hyperplasia is commonlyobserved following infection by bacteria in both post-infectious IBS andnon-post-infectious IBS.

A recent development in IBS treatment has been the low FODMAP diet. Thisdiet requires patients to restrict the intake of FODMAP carbohydrates.These are Fermentable Oligo-, Di-, Monosaccharides and Polyols which arepoorly absorbed in the proximal small intestine, osmotically active, andfermented by intestinal bacteria in which some produce hydrogen.Adherence to this diet has resulted in symptom improvements for somepatients (Staudacher et al. J. Hum. Nutr. Diet. 24, 487 (2011)).However, some of the FODMAP carbohydrates are beneficial fibres, andfoods that contain them are common, highly nutritious fruits, vegetablesand legumes.

Similarly, for lactose intolerance, there is no universally acceptedtherapy for treatment. However, existing strategies for management ofthe conditions include avoidance of lactose-containing dairy foods(milks, soft cheeses and ice creams) and consuming lactase prior to ameal containing lactose. Although restricting dietary lactose mayimprove gastrointestinal complaints, long-term effects of a diet low orfree of dairy products can be of concern, since dairy products providesa package of essential nutrients such as calcium, protein, riboflavin,vitamin A and vitamin D. Dietary calcium is an important part of therecommended daily allowance of vitamins and minerals, and for manypeople it is not possible to achieve recommended daily calcium intakeswith a dairy low or free diet. It has been observed that deficiency incalcium can lead to increased risk of osteoporosis, hypertension andpossibly cancer.

Lactase drugs are effective and are available without prescription.However, they do need to be consumed before meals containing lactose.Also, although uncommon, they may provoke serious, allergy-related sideeffects.

Therefore, there is a need for a safe, effective intervention for thetreatment of non-infectious diarrhea.

SUMMARY

This invention provides synthetic compositions comprising one or morehuman milk monosaccharides (HMSs) or one or more human milkoligosaccharides (HMOs), or both, that can be advantageously used forprophylaxis or treatment of non-infectious diarrhoea in a human, inparticular a non-infant human individual.

Accordingly: a first aspect of this invention relates to a human milkmono- or oligosaccharide or a mixture of human milk mono- and/oroligosaccharides for the prophylaxis or treatment of non-infectiousdiarrhoea in a human;

a second aspect of this invention relates to a human milk mono- oroligosaccharide or a mixture of human milk mono- and/or oligosaccharidesfor the prophylaxis or treatment of non-infectious diarrhoea in anirritable bowel syndrome patient, lactose intolerant patient and/or apatient having undergone antibiotic treatment;

a third aspect of this invention relates to a synthetic composition forthe prophylaxis or treatment of non-infectious diarrhoea in a human, thecomposition comprising an effective amount of one or more human milkmonosaccharides or one or more human milk oligosaccharides, or both;

a fourth aspect of this invention relates to a synthetic composition forthe prophylaxis or treatment of non-infectious diarrhoea in an irritablebowel syndrome patient, lactose intolerant patient and/or a patienthaving undergone antibiotic treatment, the composition comprising aneffective amount of one or more human milk monosaccharides or one ormore human milk oligosaccharides, or both;

a fifth aspect of this invention provides a method for the prophylaxisor treatment of non-infectious diarrhoea in a human, the methodcomprising administering to the human an effective amount of one or morehuman milk monosaccharides or one or more human milk oligosaccharide, orboth;

a sixth aspect of this invention provides a method for the prophylaxisor treatment of non-infectious diarrhoea in an irritable bowel syndromepatient, lactose intolerant patient and/or a patient having undergoneantibiotic treatment, the method comprising administering to the humanan effective amount of one or more human milk monosaccharides or one ormore human milk oligosaccharides, or both.

Preferably the amount of a human milk mono- and/or oligosaccharide iseffective to increase (i) the abundance, particularly the relativeabundance, of bifidobacteria, and/or (ii) the beta-galactosidaseactivity, in the gastrointestinal tract of the human. More preferably,the bifidobacteria increased is a member of the phylogeneticBifidobacterium adolescentis group, for example, Bifidobacteriumpseudocatenulatum and/or Bifidobacterium adolescentis.

The patient may have intestinal dysbiosis and/or an impaired mucosalbarrier.

Preferably, the human milk oligosaccharide is 2′-FL, 3-FL, DFL, LNnT,LNT, 3′-SL, 6′-SL or LNFP-I or a mixture thereof. For example, thecomposition can comprise a mixture of a fucosylated HMO such as 2′-FLand a non-fucosylated neutral HMO such as LNnT or LNT, or both. 2′-FLand LNnT/LNT may be present in a mass ratio of about 5:1 to 1:1; morepreferably about 4:1 to 2:1.

The synthetic composition can be a nutritional or pharmaceuticalcomposition. Preferably, synthetic composition of the invention isadministered daily. Furthermore, the synthetic composition is preferablyadministered for a period of at least one month, such as at least 2months or for a longer period of time, for example chronically on anongoing basis.

The synthetic composition may be administered to the human or patient asa daily dose of about 1 g to about 15 g such as from about 3 g to about10 g of HMSs and/or HMOs. The patient can be administered a higheramount, preferably 5 g to 10 g per day, of the HMSs and/or HMOs for aninitial treatment period, followed by a lower amount, preferably 1 g to5 g per day, for a maintenance period. The initial treatment period canbe 1 to 8 weeks. The maintenance period is at least 1 month.

Reference throughout this specification to features, advantages, orsimilar language does not imply that all of the features and advantagesthat may be realized with the present invention should be or are in anysingle embodiment of the invention. Rather, language referring to thefeatures and advantages is understood to mean that a specific feature,advantage, or characteristic described in connection with an embodimentis included in at least one embodiment of the present invention. Thus,discussion of the features and advantages, and similar language,throughout this specification may, but do not necessarily, refer to thesame embodiment.

Furthermore, the described features, advantages, and characteristics ofthe invention may be combined in any suitable manner in one or moreembodiments. One skilled in the relevant art will recognize that theinvention may be practiced without one or more of the specific featuresor advantages of a particular embodiment. In other instances, additionalfeatures and advantages may be recognized in certain embodiments thatmay not be present in all embodiments of the invention.

These features and advantages of the present invention will become morefully apparent from the following description and appended claims, ormay be learned by the practice of the invention as set forthhereinafter.

DETAILED DESCRIPTION

It has been surprisingly found that human milk monosaccharides (HMSs)and/or human milk oligosaccharides (HMOs) are able to decrease bowelmovement frequency and improve stool consistency in patients sufferingfrom non-infectious diarrhoea, particularly those who are suffering fromintestinal dysbiosis or an impaired mucosal barrier. HMSs and/or HMOspreferentially increase the abundance of bifidobacteria in thegastro-intestinal tract, metabolising carbohydrates, in particular,which escaped absorption in the small intestine, into lactate andshort-chain fatty acids, making them less osmotically active in thecolon. Further, HMSs and/or HMOs act to reduce chronic mucosalinflammation and/or repair damage to the mucosal barrier, potentiallyreducing cryptal secretion. HMSs and/or HMOs can also act on neuronallydependent gut migrating motor complexes to address disorders of gutmotility and possibly have beneficial effects on the central nervoussystems of patients. As an outcome, bowel movement frequency isdecreased and stool consistency improved.

The term “oral administration” preferably means any conventional formfor the oral delivery of a composition to a patient that causes thedeposition of the composition in the gastrointestinal tract (includingthe stomach) of the patient. Accordingly, oral administration includesswallowing of composition by the patient, enteral feeding through anaso-gastric tube, and the like.

The term “effective amount” preferably means an amount of a compositionthat provides a human milk monosaccharide or human milk oligosaccharidein a sufficient amount to render a desired treatment outcome in apatient. An effective amount can be administered in one or more doses tothe patient to achieve the desired treatment outcome.

The term “human milk monosaccharide” or “HMS” preferably means amonosaccharide found in human breast milk. Examples include sialic acidand L-fucose. In human milk, the sialic acid is N-acetylneuraminic acid.

The term “human milk oligosaccharide” or “HMO” preferably means acomplex carbohydrate found in human breast milk that can be in acidic orneutral form. More than about 200 different HMO structures are known toexist in human breast milk (Urashima et al.: Milk Oligosaccharides, NovaBiomedical Books, New York, 2011). HMOs can be backbone, fucosylated andsialylated oligosaccharides. Backbone HMOs consists of Glu, Gal andGlcNAc and are devoid of Fuc and sialic acid. Examples of backbone HMOsinclude lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT),lacto-N-neohexaose (LNnH) and lacto-N-hexaose (LNH). Fucosyl HMOs arefucosylated lactoses or fucosylated backbone HMOs such as2′-fucosyllactose (2′-FL), lacto-N-fucopentaose I (LNFP-I),lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL),difucosyllactose (DFL), lacto-N-fucopentaose III (LNFP-III),fucosyl-para-lacto-N-neohexaose (F-pLNnH), lacto-N-difucohexaose I(LNDFH-I), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V(LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I(FLNH-I), fucosyl-lacto-N-hexaose III (FLNH-III) andfucosyl-para-lacto-N-neohexaose (F-pLNnH). Sialyl HMOs are sialylatedlactoses or sialylated backbone HMOs such as3′,6-disialyllacto-N-tetraose (DSLNT), 6′-sialyllactose (6′-SL),3′-sialyllactose (3′-SL), 6′-sialyllacto-N-neotetraose (LST c),3′-sialyllacto-N-tetraose (LST a) and 6-sialyllacto-N-tetraose (LST b).HMOs containing both sialyl and fucosyl groups may be considered tobelong to either of the latter two groups. Examples for sialyl andfucosyl HMOs include disialyl-fucosyl-lacto-N-hexaose II (DSFLNH-II),fucosyl-sialyl-lacto-N-neohexaose I (FSLNnH-I),fucosyl-sialyl-lacto-N-hexaose I (FSLNH-I) and3-fucosyl-3′-sialyllactose (FSL).

The terms “microbiota”, “microflora” and “microbiome” preferably mean acommunity of living microorganisms that typically inhabits a bodilyorgan or part. The most dominant members of the gastrointestinalmicrobiota include microorganisms of the phyla of Firmicutes,Bacteroidetes, Actinobacteria, Proteobacteria, Synergistetes,Verrucomicrobia, Fusobacteria, and Euryarchaeota; at genus level themicroorganisms of Bacteroides, Faecalibacterium, Bifidobacterium,Roseburia, Alistipes, Collinsella, Blautia, Coprococcus, Ruminococcus,Eubacterium and Dorea; and at species level microorganisms ofBacteroides uniformis, Alistipes putredinis, Parabacteroides merdae,Ruminococcus bromii, Dorea longicatena, Bacteroides caccae, Bacteroidesthetaiotaomicron, Eubacterium hallii, Ruminococcus torques,Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsellaaerofaciens, Dorea formicigenerans, Bacteroides vulgatus and Roseburiaintestinalis. In some instances, the gastrointestinal microbiotaincludes the mucosa-associated microbiota, which is located in orattached to the mucus layer covering the epithelium of thegastrointestinal tract, and luminal-associated microbiota, which isfound in the lumen of the gastrointestinal tract.

The terms “irritable bowel syndrome” and “IBS” preferably mean a groupof functional bowel disorders of humans, particularly adults,characterized by one or more chronic symptoms including abdominal pain,abdominal discomfort, abdominal bloating, fatigue, and changes in bowelmovement patterns, such as patterns of loose or more frequent bowelmovements, diarrhoea and constipation, typically in the absence of anyapparent structural abnormality. There are at least three forms of IBS,depending on which symptom predominates: (1) diarrhoea-predominant(IBS-D); (2) constipation-predominant (IBS-C); and (3) IBS withalternating stool pattern (IBS-A or IBS-M). There are also variousclinical subtypes of IBS, such as post-infectious IBS (IBS-PI).

The term “bifidobacteria” means a member of the Bifidobacterium genuscommonly found in the human gastro-intestinal tract. Examples ofbifidobacteria are: Bifidobacterium longum, Bifidobacterium bifidum, andmembers of the phylogenetic Bifidobacterium adolescentis group. Innon-infant humans, bifidobacteria preferably include members of thephylogenetic Bifidobacterium adolescentis group, for example,Bifidobacterium pseudocatenulatum and/or Bifidobacterium adolescentis.

The term “synthetic composition” means a composition which isartificially prepared and preferably means a composition containing atleast one compound that is produced ex vivo chemically and/orbiologically, e.g. by means of chemical reaction, enzymatic reaction orrecombinantly. In some embodiments a synthetic composition may be, butpreferably is not, identical with a naturally occurring composition. Thesynthetic composition typically comprises one or more compounds HMSsand/or HMOs that are capable of preferentially increasing the abundanceof bifidobacteria. In some embodiments, the synthetic composition maycomprise one or more compounds or components other than HMSs and/or HMOsthat may have an effect on bifidobacteria of a human subject microbiotain vivo, e.g. non-digestible oligosaccharides or prebiotics. Also insome embodiments, the synthetic compositions may comprise one or morenutritionally or pharmaceutically active components which do not affectadversely the efficacy of the above mentioned compounds. Somenon-limiting embodiments of a synthetic composition of the invention arealso described below.

The term “relative abundance of bifidobacteria” means the abundance ofbifidobacteria relative to other genus in the microbiota of thegastro-intestinal tract.

The term “relative growth of bifidobacteria” means the growth ofbifidobacteria relative to other genus in the microbiota in thegastro-intestinal tract.

The term “non-infant human” or “non-infant” means in the present contexta human of 3 years of age and older. A non-infant human can be a child,a teenager, an adult or an elderly.

The term “enteral administration” means any conventional form fordelivery of a composition to a human that causes the deposition of thecomposition in the gastrointestinal tract (including the stomach).Methods of enteral administration include feeding through a naso-gastrictube or jejunum tube, oral, sublingual and rectal.

The term “oral administration” means any conventional form for thedelivery of a composition to a human through the mouth. Accordingly,oral administration is a form of enteral administration.

The term “about” in the present context means up to 2.5% deviation fromthe corresponded value.

The term “preferably” is used herein to indicated the best mode ofinvention, but to limit the scope of invention.

HMSs and/or HMOs for Prophylaxis or Treatment of Non-InfectiousDiarrhoea in a Human

HMSs and/or HMOs for prophylaxis or treatment of non-infectiousdiarrhoea in a human may be a single HMS, a mixture of HMSs, a singleHMO, a mixture of any HMOs or a mixture of one or more HMSs and one ormore HMOs suitable for the purpose of the invention. Preferably, the HMSis L-fucose or sialic acid, and the HMO is a fucosylated or anon-fucosylated neutral HMO. More preferably, the HMSs and/or HMOs forprophylaxis or treatment of non-infectious diarrhoea in a human is amixture of at least a first HMO, at least a second HMO and optionallyL-fucose and/or sialic, wherein the first HMO is a fucosylated neutralHMO and the second HMO is a non-fucosylated neutral HMO. Particularly,the mixture may contain a fucosylated HMO selected from the listconsisting of 2′-FL, 3-FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V,LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I, FLNH-II, FLNnH, FpLNH-I andF-pLNnH II, and a non-fucosylated HMO selected from the list consistingof LNT, LNnT, LNH, LNnH, pLNH and pLNnH. Preferably, the mixturecontains a fucosylated HMO selected from the list consisting of 2′-FL,3-FL and DFL, and a non-fucosylated HMO selected from the listconsisting of LNT and LNnT; advantageously the mixture comprises 2′-FLand LNnT and/or LNT. In some embodiments, the mixture may essentiallyconsist of two neutral HMOs, e.g. a fucosylated HMO selected from thelist consisting of 2′-FL, 3-FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V,LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I, FLNH-II, FLNnH, FpLNH-I andF-pLNnH II, and a non-fucosylated HMO selected from the list consistingof LNT, LNnT, LNH, LNnH, pLNH and pLNnH. Preferably, the mixtureessentially consists of a fucosylated HMO selected from the listconsisting of 2′-FL, 3-FL and DFL, and a non-fucosylated HMO selectedfrom the list consisting of LNT and LNnT; in one preferred embodimentthe mixture essentially consists of 2′-FL and LNnT, in another preferredembodiment the mixture essentially consists of 2′-FL and LNT.

The HMOs can be isolated or enriched by well-known processes frommilk(s) secreted by mammals including, but not limited to human, bovine,ovine, porcine, or caprine species. The HMOs can also be produced bywell-known processes using microbial fermentation, enzymatic processes,chemical synthesis, or combinations of these technologies. As examples,using chemistry LNnT can be made as described in WO 2011/100980 and WO2013/044928, LNT can be synthesized as described in WO 2012/155916 andWO 2013/044928, a mixture of LNT and LNnT can be made as described in WO2013/091660, 2′-FL can be made as described in WO 2010/115934 and WO2010/115935, 3-FL can be made as described in WO 2013/139344, 6′-SL andsalts thereof can be made as described in WO 2010/100979, sialylatedoligosaccharides can be made as described in WO 2012/113404 and mixturesof human milk oligosaccharides can be made as described in WO2012/113405. As examples of enzymatic production, sialylatedoligosaccharides can be made as described in WO 2012/007588, fucosylatedoligosaccharides can be made as described in WO 2012/127410, andadvantageously diversified blends of human milk oligosaccharides can bemade as described in WO 2012/156897 and WO 2012/156898. With regard tobiotechnological methods, WO 01/04341 and WO 2007/101862 describe how tomake core human milk oligosaccharides optionally substituted by fucoseor sialic acid using genetically modified E. coli.

Synthetic Composition Comprising HMSs and/or HMOs

The synthetic composition may comprise a single HMS, a mixture of HMSs,a single HMO, a mixture of any HMOs or a mixture of one or more HMSs andone or more HMOs suitable for the purpose of the invention. Preferably,the HMS is L-fucose or sialic acid, and the HMO is a fucosylated or anon-fucosylated neutral HMO. More preferably, the composition comprisesa mixture of at least a first HMO, at least a second HMO and optionallyL-fucose and/or sialic, wherein the first HMO is a fucosylated neutralHMO and the second HMO is a non-fucosylated neutral HMO. Particularly,the composition may contain a fucosylated HMO selected from the listconsisting of 2′-FL, 3-FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V,LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I, FLNH-II, FLNnH, FpLNH-I andF-pLNnH II, and a non-fucosylated HMO selected from the list consistingof LNT, LNnT, LNH, LNnH, pLNH and pLNnH. Preferably, the compositioncontains a fucosylated HMO selected from the list consisting of 2′-FL,3-FL and DFL, and a non-fucosylated HMO selected from the listconsisting of LNT and LNnT, advantageously the composition comprises2′-FL and LNnT and/or LNT. In some embodiments, the compositioncomprises a mixture essentially consisting of two neutral HMOs, e.g. afucosylated HMO selected from the list consisting of 2′-FL, 3-FL, DFL,LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I,FLNH-II, FLNnH, FpLNH-I and F-pLNnH II, and a non-fucosylated HMOselected from the list consisting of LNT, LNnT, LNH, LNnH, pLNH andpLNnH. Preferably, the composition comprises a mixture consisting of afucosylated HMO selected from the list consisting of 2′-FL, 3-FL andDFL, and a non-fucosylated HMO selected from the list consisting of LNTand LNnT; in one preferred embodiment the composition comprises amixture essentially consisting of 2′-FL and LNnT, in another preferredembodiment the composition comprises a mixture essentially consisting of2′-FL and LNT.

A synthetic composition of this invention comprising one or more humanmilk monosaccharides or one or more human milk oligosaccharides, orboth, can take any suitable form. For example, the composition can be inthe form of a nutritional composition which contains othermacronutrients such as proteins, lipids or other carbohydrates. Thesynthetic composition can also be a pharmaceutical composition.

Nutritional Compositions

A nutritional composition of this invention can contain sources ofprotein, lipids and/or digestible carbohydrates and can be in powderedor liquid forms. The composition can be designed to be the sole sourceof nutrition or a nutritional supplement. For IBS patients, anutritional supplement is preferred; especially a supplement which canform a meal or snack replacement. Preferably the nutritional compositionis lactose-reduced or, better yet, lactose-free. Preferably, thenutritional composition is also free from, or low in amounts of, FODMAPcarbohydrates.

Suitable protein sources include milk proteins, soy protein, riceprotein, pea protein and oat protein, or mixtures thereof. Milk proteinscan be in the form of milk protein concentrates, whey protein or casein,or mixtures of both. Soy, rice, pea and oat protein can be in the formor protein isolated. The protein can be whole protein or hydrolysedprotein, either partially hydrolysed or extensively hydrolysed. Theprotein can provide about 5% to about 50%, preferably about 10% to 30%,of the energy of the nutritional composition. The protein sourcepreferably is not a source of non-fermentable carbohydrates such aslactose. Therefore, if a milk protein is used as the protein source, themilk protein is preferably lactose-reduced or lactose-free.

The protein source can be a source of glutamine, threonine, cysteine,serine, proline, or a combination of these amino acids. The glutaminesource can be a glutamine dipeptide and/or a glutamine enriched protein.Glutamine can be included due to the use of glutamine by enterocytes asan energy source. Threonine, serine and proline are important aminoacids for the production of mucin. Mucin coats the GI tract and canreduce permeability. Cysteine is a major precursor of glutathione, whichis key for the antioxidant defences of the body.

Suitable digestible carbohydrates include maltodextrin, hydrolysed ormodified starch or corn starch, glucose polymers, corn syrup, corn syrupsolids, tapioca, sucrose, and glucose, or mixtures thereof. Generallydigestible carbohydrates provide about 35% to about 75%, preferablyabout 45% to 70%, of the energy of the nutritional composition.Preferably the digestible carbohydrate is free from lactose.

Suitable lipids include rapeseed oil, sunflower seed oil, palm oil, soyoil, milk fat, corn oil and soy lecithin. Long-chain poly unsaturatedfatty acids (LC-PUFA), especially omega-3 fatty acids such asdocosahexaenoic acid (DHA), can be included in the lipid source becausethey have anti-inflammatory properties. Suitable sources of LC-PUFA areplant oils, marine plankton oils, fungal oils, and fish oils. The lipidsource can also include medium chain triglycerides (MCT). Fractionatedcoconut oils are a suitable source of medium chain triglycerides. Thelipid source preferably provides about 5% to about 25% of the energy ofthe nutritional composition; for example, about 10% to 20%. The lipidcontent is preferably reduced because high fat diets can provoke IBSsymptoms.

The nutritional composition may also include vitamins and minerals. Ifthe nutritional composition is intended to be a sole source ofnutrition, it preferably includes a vitamin and mineral profile,preferably a complete vitamin and mineral profile. The term “complete”in the present context means a vitamin and mineral profile comprisingall vitamins and minerals essential for body function, wherein theessential vitamins includes at least 9 vitamins from the exemplary groupbelow, such as 10, 11, 12 or 13, or more, and the essential mineralsincludes at least 5 minerals from the exemplary group below, such asfrom 6 to 13 or more. Examples of vitamins include vitamins A, B-complex(such as B1, B2, B6 and B12), C, D, E and K, niacin and acid vitaminssuch as pantothenic acid and folic acid and biotin. Examples of mineralsinclude calcium, iron, zinc, magnesium, iodine, copper, phosphorus,manganese, potassium, chromium, molybdenum, selenium, nickel, tin,silicon, vanadium and boron.

The nutritional composition can also include a carotenoid such aslutein, lycopene, zeaxanthin, and beta-carotene. The total amount ofcarotenoid included can vary from about 0.001 μg/ml to about 10 μg/ml.Lutein can be included in an amount of from about 0.001 μg/ml to about10 μg/ml, preferably from about 0.044 μg/ml to about 5 g/ml of lutein.Lycopene can be included in an amount from about 0.001 μg/ml to about 10μg/ml, preferably about 0.0185 mg/ml to about 5 g/ml of lycopene.Beta-carotene can comprise from about 0.001 μg/ml to about 10 mg/ml, forexample about 0.034 μg/ml to about 5 μg/ml of beta-carotene.

The nutritional composition can also contain various other conventionalingredients such as preservatives, emulsifying agents, thickeningagents, buffers, fibres and probiotics, especially probiotics which canhelp to reduce symptoms in IBS patients (e.g. VSL#3, B. infantis 35624,B. animalis subsp. lactis BB-12, B. lactis Bi-07, L. rhamnosus GG, L.rhamnosus Lc705, L. plantarum DSM 9843, L. plantarum CECT7484, L.plantarum CECT7485, L. acidophilus NCFM, L. fermentum CECT5716, B. breveBb99, Propionibacterium freundenreichii ssp. Shermanii JS, P.acidilactici CECET7483, Streptococcus faecium),antioxidant/anti-inflammatory compounds including tocopherols,caroteinoids, ascorbate/vitamin C, ascorbyl palmitate, polyphenols,glutathione, and superoxide dismutase (melon), other bioactive factors(e.g. growth hormones, cytokines, TFG-β), colorants, flavours, andstabilisers, lubricants, and so forth.

The nutritional composition can be in the form of a soluble powder, aliquid concentrate, or a ready-to-use formulation. Various flavours,fibres and other additives can also be present.

The nutritional compositions can be prepared by any commonly usedmanufacturing techniques for preparing nutritional compositions in solidor liquid form. For example, the composition can be prepared fromvarious feed solutions. A protein-in-fat feed solution can be preparedby heating and mixing the lipid source and then adding an emulsifier(e.g., lecithin), fat soluble vitamins, and at least a portion of theprotein source while heating and stirring. A carbohydrate feed solutionis also prepared by adding minerals, trace and ultra trace minerals,thickening or suspending agents to water while heating and stirring. Theresulting solution is held for 10 minutes with continued heat andagitation before adding carbohydrates (e.g. the HMOs and digestiblecarbohydrate sources). The resulting feed solutions are then blendedtogether while heating and agitating and the pH adjusted to 6.6-7.0,after which the composition is subjected to high-temperature short-timeprocessing during which the composition is heat treated, emulsified andhomogenized, and then allowed to cool. Water soluble vitamins andascorbic acid are added, the pH is adjusted to the desired range ifnecessary, flavours are added, and water is added to achieve the desiredtotal solid level.

For a liquid product, the resulting solution can then be asepticallypackaged to form an aseptically packaged nutritional composition. Inthis form, the nutritional composition can be in ready-to-feed orconcentrated liquid form. Alternatively, the composition can be spraydried and processed and packaged as a reconstitutable powder.

When the nutritional product is a ready-to-feed nutritional liquid, thetotal concentration of HMOs in the liquid, by weight of the liquid, isfrom about 0.02% to about 2.0%, including from about 0.1% to about 1.5%,including from about 0.3% to about 1.0%. When the nutritional product isa concentrated nutritional liquid, the total concentration of HMOs inthe liquid, by weight of the liquid, is from about 0.04% to about 4.0%,including from about 0.2% to about 3.0%, including from about 0.6% toabout 2.0%.

Unit Dosage Forms

The synthetic composition can also be in a unit dosage form such as acapsule, tablet or sachet. For example, the composition can be in atablet form comprising the human milk mono-and/or oligosaccharides, andone or more additional components to aid formulation and administration,such as diluents, excipients, antioxidants, lubricants, colorants,binders, disintegrants, and the like.

Suitable diluents, excipients, lubricants, colorants, binders, anddisintegrants include polyethylene, polyvinyl chloride, ethyl cellulose,acrylate polymers and their copolymers, hydroxyethyl-cellulose,hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose,polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA),polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), or polyacrylamide(PA), carrageenan, sodium alginate, polycarbophil, polyacrylic acid,tragacanth, methyl cellulose, pectin, natural gums, xanthan gum, guargum, karaya gum, hypromellose, magnesium stearate, microcrystallinecellulose, and colloidal silicon dioxide. Suitable antioxidants arevitamin A, carotenoids, vitamin C, vitamin E, selenium, flavonoids,polyphenols, lycopene, lutein, lignan, coenzyme Q10 (“CoQIO”) andglutathione.

The unit dosage forms, especially those in sachet form, can also includevarious nutrients including macronutrients.

The unit dosage forms can be administered orally, e.g. as a tablet,capsule, or pellet containing a predetermined amount, or as a powder orgranules containing a predetermined concentration or a gel, paste,solution, suspension, emulsion, syrup, bolus, electuary, or slurry, inan aqueous or non-aqueous liquid, containing a predeterminedconcentration. Orally administered compositions can include binders,lubricants, inert diluents, flavouring agents, and humectants. Orallyadministered compositions such as tablets can optionally be coated andcan be formulated so as to provide sustained, delayed or controlledrelease of the mixture therein.

The unit dosage forms can also be administered by rectal suppository,aerosol tube, naso-gastric tube or direct infusion into the GI tract orstomach.

The unit dosage forms can also include therapeutic agents such asantiviral agents, antibiotics, probiotics, analgesics, andanti-inflammatory agents. The synthetic composition in unit dosage formmay be a pharmaceutical composition or a nutritional supplement.

Administration Dosing

For reducing symptoms of non-infectious diarrhoea in a patient, theamount of HMS(s) and/or HMO(s), preferably HMO(s), required to beadministered to the patient will vary depending upon factors such as therisk and severity of the disease, the age of the patient, the form ofthe composition, and other medications being administered to thepatient. However, the required amount can be readily set by a medicalpractitioner and would generally be in the range from about 200 mg toabout 20 g per day, in certain embodiments from about 1 g to about 15 gper day, from about 3 g to about 10 g per day, in certain embodimentsfrom about 3 g to about 7.5 g per day. An appropriate dose can bedetermined based on several factors, including, for example, body weightand/or condition, the severity of the condition, being treated orprevented, other ailments and/or diseases, the incidence and/or severityof side effects and the manner of administration. Appropriate doseranges may be determined by methods known to those skilled in the art.During an initial treatment phase, the dosing can be higher or lowerdepending upon the need to boost bifidobacteria abundance or initialtolerance to HMSs/HMOs. During a maintenance phase, the dosing can beset for chronic long term use.

The duration of the HMS/HMO administration will vary depending uponfactors such as the risk and severity of the medical condition, age, theform of the composition, the dose and other medications beingadministered. However, the duration can be readily set by a medicalpractitioner. Generally, a duration of at least a week will be requiredto sufficiently to impact symptoms. For example, the duration may be for1 to 3 months, or longer. The administration can continue chronicallyfor an indefinite period.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

EXAMPLES

Examples below are to illustrate non-limiting embodiments of theinvention.

Example 1—Human Trial in IBS-D Patients

A total of 60 male and female IBS-D patients are recruited toparticipate in the study. After a screening visit and run-in period of1-2 weeks, the patients are selected. The patients are randomized intothree groups, each of 20 patients, with two groups consuming thetreatment product and one group the placebo product for 8 weeks. Thetreatment product contains either 5 or 10 grams of a combination of2′-FL and LNnT in a 4:1 ratio while the control product contains 2 gramsof glucose. Both products are in powder form in a unit dosage container.

The patients are eligible to participate if: they are between 18 and 60years of age; meet the Rome III criteria for IBS-D; report a weeklyaverage of worst daily abdominal pain intensity score of ≥3 on a 0-10point scale; report a pain/discomfort frequency of at least 2 days aweek during screening evaluation; report at least one stool with aconsistency of Type 6 or Type 7 Bristol stool (BSS) on at least 2 daysper week. All recruited patients are able and willing to understand andcomply with the study procedures. Patients are excluded if: they haveparticipated in a clinical study one month prior to screening visit;they have abnormal results in the screening tests which are clinicallyrelevant for study participation; they are suffering for a severedisease such as malignancy, diabetes, severe coronary disease, kidneydisease, neurological disease, or severe psychiatric disease or anycondition which can confound the results of the study; used highly dosedprobiotic supplements (yoghurt allowed) for 3 months prior to the study;consumed antibiotic drugs 3 months prior to the study; consumed on aregular basis any medication that might interfere with symptomevaluation 2 weeks prior to the study; and pregnant or lactating.

At the initial visit (screening), each patient is given both written andoral information about the study and the patient is asked to sign aninformed consent form.

Patients are evaluated by a full review of clinical history, and basedon clinical symptoms, IBS-D patients are enrolled.

A blood sample for eligibility analysis is collected. A talk through ofthe electronic questionnaires (GSRS, IBS-SSS, QoL and BSFS) is performedto familiarise the patients with the electronic system, and equipmentfor faecal sampling is distributed to each patient. Patients areinstructed to keep their samples in the freezer until the next visit.

At the second visit (beginning of intervention), eligibility criteriaare checked and eligible subjects are randomised to the two arms in thetrial. Symptoms (as measured by GSRS, IBS-SSS, BSFS and QoL scales) areassessed. Trial supplementation is distributed along with instructionson use of an electronic compliance diary. The faecal samples arecollected and equipment for collecting new samples are distributed.Patients are reminded not to change their usual diet during the study.

Blood samples are collected for biomarker studies and biobanking. Theserum from the blood samples is transferred to cryotubes and stored at−80° C. The following biomarkers are measured TNF-α, IL-1β, IL-8, IL-6,IL-12, IL-10, MIP-1β, hs-CRP, lipopolysaccharide binding protein,tryptase, antiflagellin, zonulin, histamine, prostaglandin 2, andcortisol.

The faecal samples are stored at −80° C. until analysis. Microbiologicalanalysis is performed on the faecal samples using the 16S rRNA genesequence.

The study runs for 8 weeks with the patients consuming either a placeboor a treatment product daily. Patients are instructed to consume theproducts in the morning with breakfast. Compliance is monitored throughthe interactive internet enabled system. The patients also use thesystem to record:

-   -   Bristol Stool Form Scale (BSFS) information,    -   symptom information such as abdominal pain, abdominal        discomfort, abdominal cramping, abdominal bloating, and        abdominal fullness,    -   Quality of life (QoL) information,    -   IBS severity scoring system (IBS-SSS) information,    -   Additional Gastrointestinal Symptom Rating Scale (GSRS)        information.

This questionnaire includes 15 items covering five dimensions (abdominalpain, indigestion, reflux, diarrhoea, constipation) and uses aseven-graded Likert scale.

4 weeks after commencement, there is an intermediate check. A physicalexamination is done and symptoms (as measured by GSRS, IBS-SSS, BSFS andQoL scales etc.) are reassessed. Faecal samples and blood samples arecollected and analysed as before, and equipment for collection of newfaecal samples are distributed.

At the end of the intervention (8 weeks), each patient has a visit withthe medical team. A physical examination is done and symptoms (asmeasured by GSRS, IBS-SSS, BSFS and QoL scales etc.) are reassessed.Trial supplementation products are collected to check compliance. Faecalsamples and blood samples are collected and analysed as before.

The treatment patients report a reduction in pain/visceral sensitivity,a reduction in bowel movement frequency and an improvement in stoolconsistency as compared to the placebo group. The blood biomarkeranalysis indicates that the treatment patients have reduced levels ofinflammatory markers, reduced gut permeability indicating an improvedmucosal barrier, and reduced evidence of mast cell degranulation. Thefaecal analysis indicates that the treatment patients have reducedlevels of intestinal dysbiosis and a higher level of bifidobacteria;especially those of the Bifidobacterium adolescentis phylogenetic group.

Example 2—Human Trial in Lactose Intolerant Patients

A total of 60 male and female with lactose intolerance are recruited toparticipate in a randomized double-blind, parallel, placebo-controlledstudy. After a screening visit and run-in period of 1-2 weeks eligiblesubjects are randomized into two groups, each of 30 participants. Onegroup is administered a placebo product containing 2 grams of glucose,the other group is administered a treatment product containing 5 gramsof a 4:1 mass ratio mix of 2′-FL and LNnT for 30 days.

Inclusion criteria includes adults in the age of 18 to 60 years withcurrent or recent self-reported history of dairy intolerance of at least1-month duration, and with gastrointestinal symptoms after dairyconsumption. Exclusion criteria includes participation in a clinicalstudy one month prior to the screening visit; abnormal results in thescreening tests which are clinically relevant for study participation;suffering from a severe disease such as malignancy, diabetes, severecoronary disease, kidney disease, neurological disease, or severepsychiatric disease or any condition which can confound the results ofthe study; ingested anti-, pre- or probiotics 3 month prior to thestudy; consumed on a regular basis any medication that might interferewith symptom evaluation 2 weeks prior to the study; and pregnant orlactating.

At the screening visit (first visit), medical history and concomitantmedication is registered and blood samples for safety analyses arecollected. Lactose intolerance is confirmed by a 25-gram lactosechallenge, where gastrointestinal symptoms (associated with lactoseintolerance) and hydrogen production are assessed via hydrogen breathtest (HBT) for 6 hours post-lactose challenge. A positive HBT is definedas a hydrogen gas elevation of 20 parts per million (ppm) at 4time-points within 6 hours following a lactose-loading dose. A faecalsample kit is distributed, and participants are instructed to keep theircollected faecal samples in the freezer until the next visit.

At the second visit, eligibility criteria are checked and eligiblesubjects are randomised to the two arms in the trial (treatment andplacebo group). Faecal samples are collected and stored at −80° C. untilanalysis. Equipment for new faecal samples are distributed. Participantsare familiarised with an interactive internet enabled system whichrecords data daily and they are provided with either treatment orplacebo products. Subjects are asked not to change their usual diet andto avoid dairy products during the 30-day treatment period.

During the 30-day treatment period the participants consume either aplacebo or a treatment product daily. Participants are instructed toconsume the products in the morning with breakfast. Compliance ismonitored through the interactive internet enabled system. Theparticipants also use the system to record:

-   -   Bristol Stool Form Scale (BSFS) information.    -   Gastrointestinal Symptom Rating Scale (GSRS) information.    -   The GSRS questionnaire includes 15 items covering five        dimensions (abdominal pain, indigestion, reflux, diarrhoea,        constipation) and uses a seven-graded Likert scale.

At the third visit, after the 30-day treatment period, faecal samplesare collected. Additionally, the participants are challenged with25-gram lactose where lactose digestion are measured by hydrogenproduction in the HBT and evaluation of gastrointestinal symptoms(associated with lactose intolerance) is measured by participant'sself-assessment of symptoms over the 6 hours following the lactosechallenge.

After completion of the treatment period, participants are followed foran additional 30 days and instructed to reintroduce dairy foods intotheir diets. During the follow-up period, participants record BSFS andGSRS information.

At the end of the study, each participant has an exit visit with themedical team and faecal samples are collected.

Lactose digestion is measured by breath hydrogen (BH) production. BH ismeasured in parts per million (ppm) using a validated hand-held hydrogenchemical sensor (EC60 gastrolyzer, Bedfont Scientific Ltd, UnitedKingdom). Following a baseline measurement (0 hour), participants ingest25 g of lactose mixed in water. BH is then remeasured at 30 and 60 min,and at 3 hours and 6 hours after lactose challenge. The baseline valueis subtracted from readings recorded at each subsequent time interval.In general, an acceptable baseline value is 20 ppm or lower. A definitepositive value is defined as more than 20 ppm above baseline at any timepoint. Results of each BH measurement are summed to obtain a value fortotal BH.

Symptoms of lactose intolerance are recorded on a four-point Likertscale at baseline (0 h) and at 3 and 6 hours following the ingestion of25 g of lactose. Bloating, flatulence, abdominal pain and cramps areassigned using a score of 0 if there were no symptoms, 1 for mildsymptoms, 2 for moderate symptoms and 3 for severe symptoms.

To assess the microbiota profile, DNA is extracted from faecal samplesusing a 96-well PowerSoil DNA Isolation Kit (MO-BIO). A minimum of onesample-well per plate is kept empty to serve as a negative controlduring PCR. PCR is done with the forward primer S-D-Bact-0341-b-S-17 andreverse primer S-D-Bact-0785-a-A-21 with Illumina adapters attached.These are universal bacterial 16S rDNA primers, which target the V3-V4region. Following PCR program is used: 98° C. for 30 sec, 25× (98° C.for 10 s, 55° C. for 20 s, 72° C. for 20 s), 72° C. for 5 min.Amplification is verified by running the products on a 1% agarose gel.Barcodes are added in a nested PCR using the Nextera Index Kit V2(Illumina) with the following PCR program: 98° C. for 30 sec, 8× (98° C.for 10 s, 55° C. for 20 s, 72° C. for 20 s), 72° C. for 5 min.Attachment of primers is verified by running the products on a 1%agarose gel. Products from the nested PCR are normalized using theSequalPrep Normalization Plate Kit and pooled. Pooled libraries areconcentrated by evaporation and the DNA concentration of pooledlibraries is measured on a Qubit fluorometer using the Qubit HighSensitivity Assay Kit (Thermo Fisher Scientific). Sequencing is done ona MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for2×300 bp paired-end sequencing. The 64-bit version of USEARCH is usedfor bioinformatical analysis of the sequence data.

Bacterial beta-galactosidase is assessed as measures of stool enzymeactivity. Faecal beta-galactosidase is measured by adding 20 μl offaeces homogenised in buffer to 480 μg ofO-nitrophenyl-beta-D-galactopyranoside in sodium phosphate buffer (pH7.0). The reaction is allowed to proceed at 45° C. for 10 min. Sodiumcarbonate (1 M) is added to stop the reaction. Optical density at 420 nmis subsequently read and beta-galactosidase activity is reported asunits/g of stool.

The results show that oral ingestion of HMOs reduce bowel movementfrequency and improve stool consistency. The HMOs also modulate theintestinal microbiota, and specifically stimulate the growth ofbifidobacteria. The increase in abundance of bifidobacteria occurs atthe expense of Escherichia and Clostridium, which are reduced. The levelof bifidobacteria correlated positively with beta-galactosidase, andnegatively with hydrogen gas production. Additionally, the results showthat symptoms of abdominal pain, cramping, bloating, and flatulencenormally provoked by lactose consumption are improved after HMOsupplementation in individuals with lactose intolerance. Collectively,HMOs are able to increase bifidobacteria and change the intestinalenvironment, and by this, reduce gastrointestinal symptoms and inparticular diarrhoea.

Example 3—Human Trial in Antibiotic Treated Children

A total of 40 children of age 5 to 10 years are recruited to participatein the study. The children are commencing a broad spectrum, antibiotictherapy prescribed by a doctor for an infectious disorder. All recruitedchildren and their caretakers are able and willing to understand andcomply with the study procedures. Children are excluded if: they haveparticipated in a clinical study one month prior to screening visit;they are suffering from a severe disease such as gastro-intestinaldiseases, malignancy, diabetes, severe coronary disease, kidney disease,neurological disease, or severe psychiatric disease or any conditionwhich can confound the results of the study; used highly dosed probioticsupplements (yoghurt allowed) for 3 months prior to the study; consumedantibiotic drugs 3 months prior to the study, and consumed on a regularbasis any medication that might interfere with symptom evaluation 2weeks prior to the study.

At a screening visit, medical history and concomitant medication isregistered. Additionally, eligibility criteria are checked and eligiblesubjects are randomised into two groups, each of 20 children. Thetreatment period (6 weeks) is divided into two, as follows.

-   -   Period 1 (2 weeks): Group 1 (placebo), Group 2 (treatment        product).    -   Period 2 (4 weeks): Group 1 (placebo), Group 2 (treatment        product).

The treatment product contains 5 grams of a combination of 2′-FL andLNnT (4:1 mass ratio), while the placebo product contains 5 grams ofglucose. Both products are in powder form in a sachet. The products areeach administered daily as a bolus at breakfast, and diet is notcontrolled; however, the participants are asked not to change theirnormal diet over the course of the study.

At the initial visit, faecal sample kits and either treatment or placeboproducts are distributed. Each child's caretaker is instructed to keepthe faecal samples in the freezer until the next visit. The children andcaretaker are reminded not to change the children's usual diet duringthe study. A faecal sample is collected at this visit and stored at −80°C. until analysis.

The study runs for two plus four weeks with the children consumingeither placebo and/or treatment product daily. Compliance is monitoredthrough the interactive internet enabled system.

-   -   The participants also use the system to record:    -   Bristol Stool Form Scale (BSFS) information    -   Questionnaire for Paediatric Functional GI Disorders (QPFG)

The QPFG questionnaire covers dimensions such as abdominal pain anddiscomfort, bowel movement, and other gastrointestinal symptoms.

At the end of period 1, faecal samples are collected and new treatmentor placebo products are distributed. At the end of period 2 (exitvisit), each children has a visit with the medical team and faecalsamples are collected. After 6 months, faecal samples are collected at afollow up visit.

To assess the microbiota profile, DNA is extracted from the faecalsamples using a 96-well PowerSoil DNA Isolation Kit (MO-BIO). A minimumof one sample-well per plate is kept empty to serve as a negativecontrol during PCR. PCR is done with the forward primerS-D-Bact-0341-b-S-17 and reverse primer S-D-Bact-0785-a-A-21 (Klindworthet al. Nucleic Acids Res. 41, e1 (2013)) with Illumina adaptersattached. These are universal bacterial 16S rDNA primers, which targetthe V3-V4 region. The following PCR program is used: 98° C. for 30 sec,25× (98° C. for 10 s, 55° C. for 20 s, 72° C. for 20 s), 72° C. for 5min. Amplification is verified by running the products on a 1% agarosegel. Barcodes are added in a nested PCR using the Nextera Index Kit V2(Illumina) with the following PCR program: 98° C. for 30 sec, 8× (98° C.for 10 s, 55° C. for 20 s, 72° C. for 20 s), 72° C. for 5 min.Attachment of primers is verified by running the products on a 1%agarose gel.

Products from the nested PCR are normalized using the SequalPrepNormalization Plate Kit and pooled. Pooled libraries are concentrated byevaporation and the DNA concentration of pooled libraries was measuredon a Qubit fluorometer using the Qubit High Sensitivity Assay Kit(Thermo Fisher Scientific). Sequencing is done on a MiSeq desktopsequencer using the MiSeq Reagent Kit V3 (Illumina) for 2×300 bppaired-end sequencing. The 64-bit version of USEARCH (Edgar, 2013) isused for bioinformatical analysis of the sequence data.

The faecal analyses reveal that HMOs are able to prevent antibioticmitigated dysbiosis and enhance a favourable microbiota composition byincreasing the abundance of bifidobacteria and especially theBifidobacterium adolescentis phytogenic group during and post antibiotictherapy. Further the children have reduced incidence of diarrhoea.

Example 4—Nutritional Composition

A ready to feed nutritional composition is prepared from water,maltodextrin, corn syrup, sugar, milk protein concentrate, vegetable oil(canola, high oleic sunflower and corn), soy protein isolate, acaciagum, flavours, HMSs/HMOs, potassium citrate, magnesium phosphate,cellulose gel and gum, calcium carbonate, sodium ascorbate, soylecithin, choline bitartrate, calcium phosphate, alpha-tocopherylacetate, ascorbic acid, carrageenan gum, ferric pyrophosphate, flavours,sweeteners (Stevia), vitamin A palmitate, niacinamide, vitamin D3,calcium pantothenate, manganese sulphate, copper sulphate, pyridoxinehydrochloride, thiamine hydrochloride, beta carotene, riboflavin,chromium chloride, folic acid, biotin, potassium iodide, phytonadione,sodium selenite, sodium molybdate, vitamin B12.

The composition provides a nutritional supplement which is a good sourceof protein, low in fat, vitamins, minerals and antioxidants, and meetsFODMAP criteria. Further, the composition contains HMSs and/or HMOswhich are able to promote the growth of beneficial intestinal bacteriaand modulate chronic inflammation.

Example 5—Capsule Composition

A capsule is prepared by filling about 1 g of HMO into a 000 gelatinecapsule using a filing machine. The capsules are then closed. The HMOare in free flowing, powder form.

Example 6—Mucosal Barrier Function

2′-FL and LNnT are tested with respect to their ability to induce MUC2,TFF3, EIMβ, CHST5, and GAL3ST2 expression in the human LS174T cellculture model of goblet cells. The human LS174T cell line is obtainedfrom the American Type Culture Collection (ATCC). LS174T cells aremaintained in minimum essential medium (MEM) supplemented according toinstructions at 37° C. in 5% CO₂. 2′-FL and LNnT are dissolved in cellculture grade water to the required concentration. The LS174T cells aretreated with the HMO solution containing 0 or 5 mg HMO/ml.

The LS174T cells are collected and suspended in Trizol reagent and totalRNA is isolated using an RNA analysis kit (Qiagen) according to themanufacturer's instructions and the RNA isolates are quantified usingNanodrop analysis (Thermo Fisher Scientific). RNA isolates are reversetranscribed using a high capacity cDNA Reverse Transcription Kit(Applied Biosystems) to create cDNA, which is then used to assess geneexpression via quantitative RT-PCR.

For the quantitative RT-PCR, specific TaqMAN gene expression assays areobtained from Applied Biosystems, which include expression assays forMUC2, TFF3, CHST5 and GAL3ST2. Quantitative real-time PCR is performedusing TaqMAN PCR Master Mix (Applied Biosystems). Reactions are run induplicates in a 384-well plate using an Applied Biosystems 7900HT FastReal-Time PCR System. The results are analysed using SDS 2.3 softwareand calculated by delta delta Ct method. All samples are normalized toGus-β expression and fold induction is calculated over untreatedcontrols. Gene expression is expressed as fold increase compared toHMO-free control cells. The experiment is repeated three times.

The results indicate that treatment with 2′-FL and LNnT increases theexpression of the MUC2 and TFF3 genes compared to control cultures.Increased expression of goblet cell genes is specific and not universal,as evidenced by the minimal induction or lack of induction of CHST5 andGAL3 ST2, respectively. MUC2 and TFF3 are key components of the mucosalbarrier and improve mucosal barrier function.

What is claimed is:
 1. A method comprising: selecting a non-infant humanexperiencing one or more symptoms associated with diarrhea-predominantirritable bowel syndrome (“IBS-D”); selecting an effective amount of amixture of the human milk oligosaccharides (“HMOs”) 2′-fucosyllactose(2′FL) and lacto-N-neotetraose (LNnT) for administration to thenon-infant human that when administered to the non-infant human reducesa level of intestinal dysbiosis by increasing the relative abundance ofBifidobacterium adolescentis; and increasing the relative abundance ofBifidobacterium adolescentis in the gastrointestinal microbiota of thenon-infant human and reducing the one or more symptoms associated withthe IBS-D in the non-infant human by administering to the non-infant adaily dose of the selected effective amount of the mixture of 2′FL andLNnT.
 2. The method of claim 1, further comprising improving an impairedmucosal barrier in the non-infant human by administering to thenon-infant human the selected effective amount of the mixture of 2′FLand LNnT.
 3. The method of claim 1, wherein reducing the one or moresymptoms associated with IBS-D comprises reducing bowel movementfrequency and improving stool consistency.
 4. The method of claim 1,further comprising reducing levels of an inflammatory marker in thenon-infant human by administering to the non-infant human the selectedeffective amount of the mixture of 2′FL and LNnT.
 5. The method of claim1, further comprising administering with the mixture of 2′FL and LNnT,one or more additional HMOs selected from the group consisting of3-fucosyllactose (3FL), difucosyllactose (DFL), lacto-N-tetrose (LNT),3′-sialyllactose (3′SL), 6′-sialyllactose (6′SL), lacto-N-fucopentaose I(LNFP-I), and combinations thereof.
 6. The method of claim 1, whereinthe mass ratio of the 2′FL to the LNnT is from about 4:1 to 2:1.
 7. Themethod of claim 1, wherein the selected effective amount of theadministered mixture of 2′FL and LNnT is from about 3 g to about 7.5 gper day.
 8. The method of claim 1, further comprising reducing levels ofEscherichia and Clostridium in the non-infant human.
 9. A methodcomprising: selecting a non-infant human experiencing lactoseintolerance and one or more symptoms associated with the lactoseintolerance, the symptoms selected from diarrhea, abdominal pain,cramping, bloating, and flatulence; selecting an amount of a mixture ofthe human milk oligosaccharides (“HMOs”) 2′-fucosyllactose (2′FL) andlacto-N-neotetraose (LNnT) effective for increasing the relativeabundance of Bifidobacterium adolescentis in the gastrointestinalmicrobiota of the non-infant human; and increasing the relativeabundance of Bifidobacterium adolescentis in the gastrointestinalmicrobiota of the non-infant human and reducing at least one of the oneor more gastrointestinal symptoms of the lactose intolerance byadministering to the non-infant human a daily dose of the selectedeffective amount of the mixture of 2′FL and LNnT.
 10. The method ofclaim 9, wherein: after an initial treatment period, the non-infanthuman reintroduces consumption of dairy foods; and administering themixture reduces the likelihood of the symptoms of lactose intoleranceoccurring in the non-infant human after the initial treatment period.11. A method comprising: selecting a non-infant human commencing anantibiotic therapy; selecting an amount of a mixture of the human milkoligosaccharides (“HMOs”) 2′-fucosyllactose (2′FL) andlacto-N-neotetraose (LNnT) effective for increasing the relativeabundance of Bifidobacterium adolescentis in the gastrointestinalmicrobiota of the non-infant human; and increasing the relativeabundance of Bifidobacterium adolescentis in the gastrointestinalmicrobiota of the non-infant human and reducing the likelihood of thenon-infant human experiencing gastrointestinal dysbiosis, diarrhea, orboth, associated with the antibiotic therapy by administering to thenon-infant human a daily dose of the selected effective amount of themixture of 2′FL and LNnT.
 12. The method of claim 11, further comprisingreducing levels of Clostridium in the non-infant human.
 13. The methodof claim 12, further comprising reducing the likelihood of thenon-infant human experiencing diarrhea associated with the Clostridiumduring the antibiotic therapy.
 14. The method of claim 12, furthercomprising reducing the likelihood of the non-infant human experiencingdiarrhea associated with the Clostridium during a post antibiotictherapy period of up to four weeks.
 15. The method of claim 9, furthercomprising reducing hydrogen gas production in the gastrointestinaltract of the non-infant human.
 16. The method of claim 9, furthercomprising increasing the beta-galactosidase activity in thegastrointestinal tract of the non-infant human.